Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4R KO mice after ischemic stroke

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-seq technology to identify the gene expression profiles, pathway analysis, and qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 KO and littermate WT mice to ischemic stroke. After 3 days, mice were sacrificed, total RNA was isolated using Trizol, and subjected to RNA-seq and Nanostring-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify mRNA transcript expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs WT tissue after stroke. Out of these DEGs, 1920 gene were downregulated, and 325 genes were upregulated in KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (IL-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.


Introduction
Ischemic stroke is the leading cause of disability in the United States. It is a vascular accident that occurs, often without warning, when there is a blockage of cerebral blood ow, and results in damage to the surrounding brain tissue [1]. Despite recent advances, interventions to reduce damage and increase recovery after stroke are limited.
Therapies, such as physical, occupational, and speech, are the only currently viable options available for post-stroke recovery [2]. This prompted us to explore pathophysiological events and mechanisms of injury to identify novel drug targets and their downstream pathways. Acute ischemic stroke triggers a series of events: rapid activation of resident microglia, release of chemokines and cytokines in the circulation, mobilization of immune cells from bone marrow, and the in ltration of circulating immune cells in the brain, primarily to phagocytose dead cells [3]. However, excessive in ltration of these immune cells into the ischemic region creates unwanted in ammation during the acute stroke phase by releasing in ammatory cytokines which leads to secondary damage after stroke, thus delaying recovery [4]. Among in ltrating cells, migration of peripheral myeloid cells (i.e., monocytes and neutrophils) to the CNS and their temporal contribution to ischemic injury is a well-known immune response. Peripheral (e.g., monocytes/macrophages) and CNS-resident (microglia) myeloid cells contribute to both acute injury and functional recovery after stroke [5]. Despite the crucial participation of myeloid cells in stroke outcomes, approaches to regulate myeloid cell response remain unsuccessful due to their complex function including both cerebroprotective and damaging roles in stroke.
The effects of aging on the immune system occur at multiple levels, including reductions in lymphoid and increases in myeloid progenitor cells by hematopoietic stem cells [6]. Despite increases in their total systemic number, myeloid cells from aged mice are not as robust as their young counterparts and show diminished phagocytosis and reduced production of cerebroprotective secretory molecules; however, aged immune cells show increased secretion of in ammatory mediators [7]. We previously showed that aged mice exhibit marked differences in the composition of circulating and in ltrating leukocyte recruitment after stroke [7]. Aged microglia also show increased sensitivity to ATP [8]. Therefore, we hypothesize that P2X4R activation in aged mice with ischemic stroke may show more pronounced neuroin ammatory cytokine/chemokine gene expression as compared to young mice. We also hypothesize that there will be some age-independent differentially expressed genes (DEGs) contributing to cerebroprotective or cerebrodetrimental effects. We will test this hypothesis by directly comparing P2X4R response in young and aged mice after stroke.
Our previous work revealed that the acute overactivation of P2X4, a purinergic receptor for ATP, exacerbated postischemic in ammation and hampered recovery [9]. Using short-term pharmacological blockade and genetic deletion, we showed that P2X4R can be a potential drug target for the treatment of acute ischemic stroke [10]. In the CNS, besides its acute neuroin ammatory role, P2X4 also plays a signi cant role in modulating synaptic transmission and communication between neurons and neighboring glial cells [11]. We and others have shown that myeloid cells express the highest amount of P2X4R and can modulate neuroin ammation mediated by chemokines, cytokines, and secondary activation of other immune cells such as T cells [10,12,13]. However, its molecular mechanism remains unknown. Here we used bulk RNAseq analysis using ischemic brain tissue from P2X4R KO and WT mice to identify common pathways and molecular mediators of damage and recovery. Transcriptomic analysis, which involves measuring the expression levels of thousands of genes simultaneously, provides a comprehensive view of the molecular changes that occur after ischemic stroke and allows for the identi cation of DEGs that may be involved in the pathophysiology of the disease [14].
The use of transcriptomic analysis in this study can help identify the speci c genes and pathways that are affected by the knockout of P2X4R and can shed light on the potential mechanisms by which P2X4R may be involved in ischemic stroke. This information can then be used to develop targeted therapies that speci cally modulate the expression or activity of these genes and pathways.

Animals and diets
We used both young (2-3-month-old) and aged (16-18-month old) Global P2X4R knockout (KO) and littermate control (WT) mice of both sexes, generated in-house at the UConn Health animal facility. Mice were fed a standard chow diet and water ad libitum. Standard housing conditions were maintained at a controlled temperature with a 12h light/dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of University of Connecticut Health and conducted in accordance with the U.S. National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Experimental design and stroke surgery:
A total of 22 young and 26 aged mice (containing both males and females) were used in this study. Out of 48 mice, 32 mice ( were subjected to a transient right middle cerebral artery occlusion (MCAo) for 60 min followed by 3 days of reperfusion as described previously [9,10], while 10 mice (4 young and 6 aged) were subjected to sham surgery and the remaining 6 naïve young mice were used for bone marrow cell isolation. For MCAo surgery, a midline ventral neck incision was made to expose the right common carotid artery which was followed by isolation of both the external and internal carotid artery. A unilateral right MCAo was achieved by inserting a 6.0 silicone rubber-coated mono lament (size 602145/602245/602345; Doccol Corporation, Sharon, MA) about 10-11 mm away from the bifurcation point of the internal carotid artery through an external carotid artery stump. After 60 mins of occlusion, mono lament was removed to perfuse the brain tissue. Rectal temperatures were monitored and maintained at 37 ± 0.5°C with the help of a heating pad. We used laser Doppler owmetry (DRT 4/Moor Instruments Ltd, Devon, UK) to measure cerebral blood ow and to con rm occlusion (reduction to 15% of baseline cerebral blood ow). Four aged mice were died after stroke so nal data includes total 44 mice.

Brain tissue isolation and total RNA extraction
Three days after MCAo, all the mice were sacri ced and perilesional prefrontal cortex tissue was isolated from the ischemic hemisphere to extract total RNA using Trizol. We used 500 ng and 200 ng RNA from each sample for RNAseq sample preparations (Illumina Hi Seq, Yale Center for Genome Analysis, New Haven, CT) and nano string platform (IDDRC molecular genetics core at BOSTON CHILDREN'S HOSPITAL, Boston, MA), respectively.

Bone marrow derived monocyte (BMDM) isolation:
After sacri ce, P2X4R KO and littermate WT mice (8-10 weeks old) were sacri ced and bones (femur and tibia) were harvested after careful dissection in aseptic conditions. The harvested bone was cut from both ends and the internal lumen was ushed with RPMI media into a collecting tube. Following RBC lysis, single cell suspension was poured into a culture dish to grow for 10-14 days. Bone marrow-derived cells were fed with GM-CSF (10 ng/ml) to differentiate into macrophages. Once macrophage cultures were established, a comparative study was conducted between macrophages of P2X4R KO and WT groups under de ned conditions. At the end of the experiment, the harvested cells were used for total RNA isolation using Trizol.

Quality Control
Total RNA quality was determined by estimating the A260/A280 and A260/A230 ratios by nanodrop. RNA integrity was determined by running an Agilent Bioanalyzer gel, which measures the ratio of the ribosomal peaks.

RNA-Seq Library Prep
mRNA was puri ed from approximately 500 ng of total RNA with oligo-dT beads and sheared by incubation at 94 o C.
Following rst-strand synthesis with random primers, second strand synthesis was performed with dUTP for generating strand-speci c sequencing libraries. The cDNA library was then end-repaired, A-tailed, adapters were ligated, and second-strand digestion was performed by Uracil-DNA-Glycosylase. Indexed libraries that met appropriate cut-offs for both were quanti ed by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥ 0.5 ng/µl were used for sequencing.

Flow Cell Preparation and Sequencing
Sample concentrations were normalized to 10 nM and loaded onto Illumina HiSeq4000 ow cells at a concentration that yields 300 million passing lter clusters per lane. Samples were sequenced using 100 bp paired-ends sequencing according to Illumina protocols. The 8 bp index was read during an additional sequencing read that automatically follows the completion of read 1. Data generated during sequencing runs were simultaneously transferred to the YCGA high-performance computing cluster. A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time.

Primary RNA-Seq Analysis and Storage
Signal intensities were converted to individual base calls during a run using the system's Real Time Analysis (RTA) software. Base calls were transferred from the machine's dedicated personal computer to the High-Performance Computing cluster at Yale Center for Research Computing (New Haven, CT) via a 1 Gigabit network mount for downstream analysis.

Secondary RNA-Seq Data Analysis
Our analysis was carried out as follows. The reads were trimmed for quality using custom scripts. Minimum length accepted was 45 bases. The trimmed reads were then aligned to the mm10 reference genome using gencode annotation (Frankish et al., 2019) using HISAT2 for alignment, and StringTie for transcript abundance estimation (Kim et al., 2015). The generated counts were processed with DESeq2 (Love et al., 2014) in R to determine signi cantly expressed genes. Internally, the DESEq2 uses Wald's test for each gene to determine if the log fold change is statistically signi cant. Wald test itself is a variation of χ 2 test (This is one reason why one needs to use the multiple hypothesis testing correction to determine truly statistically signi cant genes). For the current analysis, we used adjusted p-value < 0.05 (effectively accepting 5% false discovery rate). The adjustment applied to the pvalue is one of the mildest corrections.  (Table 1). Data was normalized as per manufacturer's instructions by using the nSolver Software (NanoString Technologies) and used for differential expression as in [10].
We performed RNA-sequencing (RNA-seq) using total RNA isolated from perilesional ipsilateral cortex of young P2X4R KO and WT mice. After quality ltering and normalizing the raw sequencing data, we identi ed DEGs based on the following criteria: fold change > 2 or < 0.5, and false discovery rate (FDR) < 0.05. To understand the molecular and cellular impacts of these DEGs, we analyzed the top 300 genes (consisting of both up-and down-regulated genes) among KO vs. WT mice after stroke using GO and IPA analysis. Among the top gene candidates, we validated about 30 biologically relevant genes using nanostring technology.

GO analysis
Using the top 300 DEGs, we identi ed 17 total signi cantly enriched GO terms ( Table 2) with 11 related to biological process and 6 related to molecular function. The top hit in the biological process and molecular function category were related to extracellular matrix, cellular origination, angiogenesis, and in ammatory response function (Fig. 1).  (Fig. 2). A negative z-score (Blue bars) indicates pathway inhibition in P2X4R KO vs. WT. The p-value, calculated with the Fischer's exact test, indicates the likelihood that the association between a set of genes in our dataset and a biological function is signi cant. Analysis of the top 16 pathways identi ed that P2X4R regulates 12 main functions connected to several molecular pathways (Fig. 1). Among these, neuroin ammatory signaling pathways and immune cell activation were prominent.

qPCR validation of the top 30 biologically relevant genes.
Hierarchical clustering analysis of the top 30 biologically relevant DEGs showed (Fig. 3) that most differentially regulated genes were related to extracellular matrix, cell-cell adhesion and immunomodulatory pathways. Further, we identi ed several signi cantly modulated in ammatory cytokines or chemokines in young brain tissue (Fig. 4a) and BMDMs (Fig. 4b) and aged brain tissue (Fig. 4c). We identi ed many novel downstream targets of P2X4R in the transient MCAo model of ischemic stroke.

Discussion
Our previous study demonstrated that Global P2X4R KO protects mice from further damage after ischemic stroke, yet the underlying molecular networks associated with P2X4R in the neuro-in ammatory response to cortical injury due to ischemic stroke remain unexplored [9]. We used next-generation RNAseq to analyze gene expression pro les generated from P2X4R KO and littermate WT mice to identify potential biological and molecular function pathways and related signaling networks in the absence of P2X4R during acute neuro-in ammation after ischemic stroke injury using transient MCAo mouse model. Here, we have identi ed many up-and down-regulated genes, revealing that the absence of P2X4R has widespread effects after ischemic stroke.
Gene ontology (GO) enrichment analysis of DEGs suggest many biological processes such as cellular integrity, organization, positive regulation of angiogenesis, and in ammatory response modulation are among the top biological processes which are affected by the absence of P2X4R. The dependence of these biological processes on P2X4R was further supported by molecular function pathways by GO analysis and canonical pathways by IPA.
These two independent analysis platforms suggest that immune cell activation/neuroin ammation and leukocyte extravasation pathways/function are dominantly altered by the absence of P2X4R. We and others have previously shown that P2X4R activation exacerbates in ammation and acute ischemic injury [3,510]. We previously showed that short-term blockade of P2X4R reduces acute ischemic injury by reducing neuroin ammation and by reducing leukocyte in ltration into the brain after stroke [10]. These data also suggest that P2X4R plays a signi cant role in blood-brain barrier (BBB) integrity, either by activation of P2X4R on myeloid cells or its interaction with endothelial cells. However, it is not yet clear if these effects on BBB permeability are mediated by myeloid or endothelial P2X4R activation [10,11]. Our qPCR data with BMDM cells suggest that the absence of P2X4R increases transcripts like COL1A2, LOX, and LOXL2. These genes are involved in rebuilding of ECM by cross-linking collagen and elastin bers, which can improve the brain's structural integrity and support its recovery following a stroke. It is well established that ECM integrity is compromised after stroke [17]. These ndings support the notion that myeloid P2X4R activation might play a major role in ECM degradation and cell to cell adhesion, thus may affect BBB integrity.
Our qPCR data validated data from young, stroked mice showing that the absence of P2X4R reduced several in ammatory transcripts like P2X7R, Tgfb1, and MRC1. Among them, MRC1, which is a member of the C-type lectin (CLEC) family of mannose receptor, has diverse roles and can bind and internalize a variety of endogenous and pathogen-associated ligands [18]. Because of these properties, its role in endocytosis as well as antigen processing and presentation has been studied intensively and is consistent with the role of P2X4R in endocytosis [19]. Our data suggest that the endocytosis roles of P2X4R might be mediated by MRC1. Besides endocytosis, it can also directly in uence the activation of various immune cells by its expression during ischemic stroke [20]. Both ischemic brain tissue in young mice and BMDM data show signi cant decrease in MRC1 transcript in P2X4R KO group, indicating that cerebroprotective effects of P2X4R blockade might be mediated by MRC1-mediated immune cell activation.
MRC1 also has a regulatory effect on the induction of immune responses which are distinct from antigen uptake and presentation, speci cally it regulates T-cell activation [21]. This regulatory effect of the MRC1 was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated protein. Given that the mannose receptor plays an important role in phagocytosis and clearance of cellular debris [22], it will be worthwhile to study how P2X4R blockade affects phagocytic uptake and T-cell activation after ischemic stroke. Our data suggest that MRC1-P2X4R may play important diverse roles during acute ischemic injury. Interestingly, our data found an age-independent decrease in expression of transcript Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2a) in P2X4R KO mice after stroke. This nding suggests that the effects MRC1 on T cells might be mediated by CTLA-2a in a P2X4R-dependent manner persisting in aging.
CTLA-2a is a cysteine proteinase inhibitor which was originally discovered in mouse-activated T cells and mast cells.
Previously it has been shown that T cell activation is detrimental during acute ischemia, and lymphocyte-de cient mice are protected in models of focal ischemia as discussed in detail in [3]. The cytotoxic activity of T cells may be related to innate functions of T cells. Further, P2X4R activation increases T cell activation and their migration to injured tissue [13]. This evidence suggests that loss/blockade of P2X4R in the brain reduces CTLA-2a expression on T cells to inhibit their activation and migration to ischemic tissue, which is consistent with IP analysis data. CTLA2a was reduced both in young and aged mice after stroke and stroke mainly occurs in aging population. This observation suggests that CTLA-2a may be a downstream target of P2X4R and can be a potential therapeutic target to treat ischemic stroke. Similar to CTLA-2a, we also found reduced expression of IL-6 after stroke in both young and aged P2X4R KO mice. Although the pro-in ammatory role of IL-6 is well-de ned after acute stroke injury, reduced IL-6 levels may indicate cerebroprotective effects of P2X4R KO during early stroke injury timepoints. Although the exact mechanism of how P2X4R activates IL-6 is not clear, we and others have shown that pro-in ammatory effects of P2X4R activation might indirectly contribute in stroke injury [23].
Taken together, our RNAseq data support the hypothesis that P2X4R modulates BBB integrity, in ammatory response, and immune cell activation and in ltration. This data also identi es two novel potential downstream targets of P2X4R during ischemic stroke: MRC1 and CTLA-2a. Data from this work support not only a key role for P2X4R in modulating the complex networks of cell death and the immune response in myeloid cells after ischemic stroke but also suggest a new role in T cell activation and a potential mechanism for this T-cell activation.   Heat map of top 30 DEGs hierarchical clustering analysis. The right 3 columns represent WT controls, and the left 3 columns represent P2X4R KO mice. Each row represents a single gene. The color change from yellow to black represents log (TPM) value ranging from high to low. This heat map represents expression data of top 30 immune related genes validated by nano string panel using mouse brain tissue of young, stroked mice (n=3/ genotype).

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